Ion channel clustering in myelinated axons is essential for proper nervous system function. Ion channels are clustered at nodes of Ranvier through neuron-glia interactions. However, the mechanisms responsible for channel clustering remain poorly understood. Recent studies suggest that multiple mechanisms may contribute to node formation. Among these, the axonal submembranous cytoskeleton comprised of ankyrins and spectrins has been proposed to be key components. In particular, nodes of Ranvier themselves are enriched with ankyrinG (ankG) and ?IV spectrin; ankG is thought to bind directly to the Na+ and K+ channels, and then link to the actin cytoskeleton through ?IV spectrin. At paranodes, both ankyrinB and ?II spectrin are clustered, although their functions at paranodes remain unknown. Paranodes are also thought to function as a paranodal diffusion barrier and a second mechanism to mediate ion channel clustering at nodes, although the mechanisms responsible for this barrier function remain unknown. Furthermore, a major impediment to elucidating the function of paranodes is the relatively few proteins that have been identified at this site. In this proposal we will seek to determine the function of the nodal and paranodal cytoskeletons in node of Ranvier assembly and maintenance. We will do this using three new mouse models that utilize Cre-Lox technology to control the temporal and spatial (cell-type specific) expression of ankG, ankB, and ?II spectrin. We will silence expression of these proteins in peripheral sensory neurons, in retinal ganglion cells, and in myelinating glia during both development and in adults. Finally, we will use proteomic methods to identify new paranodal proteins, and then seek to determine their functions. PUBLIC HEALTH RELEVANCE: Nodes of Ranvier are required for proper nervous system function, and many diseases or injuries disrupt nodes. Therapies for nervous system diseases will require a detailed understanding of how nodes form. We propose that the cytoskeleton consisting of ankyrins and spectrins plays important roles in node assembly and maintenance. We have developed a variety of mutant mouse models that will allow us to test this idea.